IL-17E Human, HEK

What is the structural composition of recombinant human IL-17E?

Recombinant human IL-17E is a disulfide-linked homodimer. When produced in E. coli expression systems, it forms a non-glycosylated protein containing two 145-amino acid chains (with an N-terminal methionine for each chain, totaling 146 amino acids per chain) and has a predicted molecular mass of approximately 33.7 kDa . The human cell-expressed version encompasses amino acids Tyr33-Gly177 of the native protein . The disulfide linkages are critical for proper folding and biological activity.

How should researchers optimally reconstitute and store IL-17E for experimental use?

For optimal reconstitution of lyophilized IL-17E:

• With carrier protein: Reconstitute at 100 μg/mL in 4 mM HCl containing at least 0.1% human or bovine serum albumin

• Carrier-free versions: Reconstitute at 100 μg/mL in 4 mM HCl

Storage recommendations:

• Store at -20°C upon receipt

• Use a manual defrost freezer

• Avoid repeated freeze-thaw cycles that can degrade the protein and reduce activity

• Working aliquots should be prepared to minimize freeze-thaw cycles

What is the biological activity range of human IL-17E in functional assays?

Human IL-17E typically demonstrates biological activity in the range of 0.25-1.5 ng/mL in standard functional assays . In HEK-Blue IL-17 reporter systems, the detection range for human IL-17E is 1-100 ng/ml . The dose-response curve tends to plateau above 100 ng/ml, indicating receptor saturation. Researchers should construct full dose-response curves when first establishing assays with a new batch of IL-17E to determine optimal working concentrations.

What are the key signaling pathways activated by IL-17E?

IL-17E activates multiple signaling pathways:

• NF-κB and AP-1 pathways: Primary pathways activated upon IL-17E binding to its receptor

• JNK and p38 MAPK: These intracellular pathways differentially regulate the upregulation of IL-17E receptors and release of cytokines/chemokines

• Promotes Th2 cytokine production: IL-4, IL-5, IL-13

• Suppresses Th1/Th17 cytokines: IFN-gamma, IL-12, IL-23, IL-17A, IL-17F

How does IL-17E differ functionally from other IL-17 family members in immunological contexts?

While the IL-17 family generally promotes inflammatory responses, IL-17E stands apart with distinct immunoregulatory functions:

This functional divergence makes IL-17E a therapeutic target in allergic disorders, while other family members are targeted in autoimmune conditions .

What protocol modifications are needed when studying IL-17E proteolytic activation by MMP-7?

When investigating MMP-7-mediated activation of IL-17E, researchers should consider:

• Pre-incubation conditions: Recombinant IL-17E should be pre-incubated with purified MMP-7 at a 1:10 enzyme-to-substrate ratio for 1-2 hours at 37°C

• Buffer composition: Use a buffer containing 10 mM CaCl₂ to ensure optimal MMP-7 activity

• Verification of cleavage: Confirm processing by SDS-PAGE or Western blotting to detect the cleaved form

• Functional comparison: Compare biological activity between cleaved and intact IL-17E by measuring:

  • Receptor binding affinity to IL-17RB

  • Induction of Th2 cytokines in target cells

This approach will help determine how proteolytic processing enhances IL-17E binding to IL-17RB and strengthens Th2 cytokine induction, as observed in airway allergic responses .

How can researchers address conflicting IL-17E expression data in tissue-specific studies?

Discrepancies in IL-17E expression data can arise from several methodological factors:

• Detection technique sensitivity:

  • qPCR typically offers greater sensitivity than protein-based methods

  • Use digital droplet PCR for low-abundance transcripts

  • Validate with multiple antibody clones for protein detection

• Cell isolation procedures:

  • Enzymatic digestion can alter surface receptor expression

  • Compare mechanical versus enzymatic isolation methods

  • Use gentle isolation protocols to preserve cytokine production capacity

• Timing considerations:

  • IL-17E expression can be transient during immune responses

  • Perform time-course experiments (6h, 12h, 24h, 48h, 72h)

  • Consider diurnal variations in expression

• Cross-validation approaches:

  • Triangulate findings using orthogonal methods (e.g., RNA-seq, proteomics, and functional assays)

  • Single-cell approaches can resolve heterogeneous expression patterns

  • In situ techniques preserve spatial context

When conflicting data emerge, systematic comparison of methodologies and detailed reporting of experimental conditions can help resolve apparent contradictions .

What is the optimal protocol for using HEK-Blue IL-17 cells to detect IL-17E bioactivity?

For optimal detection of IL-17E bioactivity using HEK-Blue IL-17 reporter cells:

Day 1:

• Prepare HEK-Blue IL-17 cell suspension at ~280,000 cells/ml in test medium

• Add 20 μl of IL-17E sample per well in a flat-bottom 96-well plate

• Include positive control (recombinant human IL-17A at 10 ng/ml final concentration) and negative control (recombinant human TNF-α at 10 ng/ml)

• Add 180 μl of cell suspension (~50,000 cells) per well

• Incubate overnight at 37°C in 5% CO₂

Day 2:

• Prepare QUANTI-Blue Solution according to manufacturer's instructions

• Transfer 20 μl of induced cell supernatant to a new flat-bottom 96-well plate

• Add 180 μl of resuspended QUANTI-Blue Solution

• Incubate at 37°C for 30 min to 3 hours

• Measure SEAP levels using a spectrophotometer at 620-655 nm

The detection range for human IL-17E is 1-100 ng/ml, with a typical dose-response curve showing sensitivity comparable to that of IL-17A .

How can researchers optimize HEK-Blue IL-17 cells for high-throughput screening of IL-17E inhibitors?

For adapting HEK-Blue IL-17 cells to high-throughput screening of IL-17E inhibitors:

• Assay miniaturization:

  • Reduce volumes to 384-well format (10 μl cells + 5 μl compound + 5 μl IL-17E)

  • Maintain cell density at 50,000 cells per well

  • Use automated liquid handling for consistent results

• Screening optimization:

  • Run Z'-factor determination using known inhibitors

  • Establish staggered addition protocol (pre-incubate compounds with IL-17E for 30 min)

  • Include concentration-response curves of reference inhibitors

• Data normalization:

  • Include both positive (maximal stimulation) and negative (no stimulation) controls on each plate

  • Calculate percent inhibition relative to controls

  • Apply robust statistical methods (e.g., B-score) to account for plate position effects

• Counter-screening:

  • Test hits against alternative activators of NF-κB/AP-1 pathways

  • Evaluate cytotoxicity using parallel viability assays

  • Confirm specificity by testing against other IL-17 family members

This approach enables efficient screening of large compound libraries while minimizing false positives and identifying selective IL-17E inhibitors .

What methodological approaches can address cross-reactivity when studying both human and mouse IL-17E in the same system?

When working with both human and mouse IL-17E in the same experimental system:

• Receptor expression analysis:

  • HEK-Blue IL-17 cells respond to both human and mouse IL-17E

  • Characterize relative expression levels of IL-17RA and IL-17RB receptors by flow cytometry

  • Consider species-specific blocking antibodies to isolate responses

• Species-specific dose-response assessment:

  • Determine EC₅₀ values for both human and mouse IL-17E

  • Human IL-17E typically shows higher potency in human cells

  • Quantify cross-reactivity ratios to calibrate mixed-species experiments

• Selective inhibition strategies:

  • Use species-selective blocking antibodies

  • Design species-specific siRNA knockdown of receptor components

  • Consider CRISPR editing to create species-selective reporter cells

• Data interpretation:

  • Always run parallel species-specific controls

  • Account for differences in receptor binding kinetics

  • Be aware that HEK-Blue IL-17 cells show stronger responses to human IL-17E than mouse IL-17F or IL-17A

This systematic approach allows researchers to dissect species-specific contributions in complex biological systems.

How should researchers design experiments to study IL-17E's differential effects on various cell types?

When investigating IL-17E's cell type-specific effects:

• Target cell panel preparation:

  • Include epithelial cells (airway, intestinal, skin)

  • Test multiple immune cell types (T cells, eosinophils, basophils, dendritic cells)

  • Compare primary cells with relevant cell lines

  • Consider tissue-resident vs. circulating cell populations

• Expression verification:

  • Confirm IL-17 receptor expression (IL-17RA/IL-17RB heterodimer) on each cell type

  • Quantify receptor levels by flow cytometry or qPCR

  • Assess expression of downstream signaling components (ACT1, TRAF6)

• Multiparameter response measurement:

  • Cytokine/chemokine production (ELISA, multiplex assays)

  • Transcriptional changes (RNA-seq, targeted qPCR panels)

  • Signaling pathway activation (phospho-flow, Western blot for NF-κB, AP-1, MAPK)

  • Functional changes (migration, proliferation, survival)

• Context-dependent modulation:

  • Test IL-17E effects in various inflammatory milieus

  • Examine synergy with allergic mediators (TSLP, IL-33)

  • Compare effects in homeostatic vs. inflammatory conditions

This comprehensive approach will reveal the tissue- and cell-specific impact of IL-17E across multiple biological contexts .

What are the methodological considerations for studying IL-17E in airway inflammation models?

For investigating IL-17E in airway inflammation models:

• Sample processing protocol optimization:

  • Bronchoalveolar lavage (BAL): Process immediately and use protease inhibitors

  • Lung tissue: Standardize dissociation methods to preserve cell viability

  • Epithelial brushings: Optimize RNA/protein extraction from limited material

• Timing considerations:

  • Early phase (0-6h): Focus on epithelial activation and initial cytokine release

  • Intermediate phase (24-48h): Measure immune cell recruitment and activation

  • Late phase (72h+): Assess tissue remodeling and chronic inflammation markers

• Cell-specific analysis:

  • Flow cytometric identification of IL-17E-producing cells

  • Single-cell RNA-seq to capture heterogeneous responses

  • In situ hybridization to maintain spatial context of expression

  • Cell-specific knockout models to determine cellular sources

• Functional readouts:

  • Airway hyperresponsiveness measurements (methacholine challenge)

  • Mucus production (PAS staining, MUC5AC quantification)

  • Eosinophil and Th2 cell infiltration

  • Local cytokine profiles (IL-4, IL-5, IL-13)

These methodological considerations will help researchers accurately characterize IL-17E's role in allergic airway inflammation and asthma pathophysiology .

How can researchers effectively study the therapeutic potential of IL-17E antagonism?

To investigate IL-17E antagonism as a therapeutic strategy:

• Antagonist selection and characterization:

  • Neutralizing antibodies against IL-17E

  • Soluble receptor constructs (IL-17RB-Fc)

  • Small molecule inhibitors of IL-17E/receptor interaction

  • Verify specific binding and neutralizing capacity in vitro before in vivo studies

• Preclinical model selection:

  • Acute vs. chronic allergen models

  • House dust mite vs. OVA-induced inflammation

  • Models with established epithelial barrier dysfunction

  • Humanized mouse models for increased translational relevance

• Intervention timing optimization:

  • Prophylactic (before allergen challenge)

  • Early intervention (at symptom onset)

  • Therapeutic (during established disease)

  • Comparative efficacy at different disease stages

• Comprehensive outcome assessment:

  • Primary outcomes: AHR, inflammation scores, symptom indices

  • Mechanistic outcomes: Th2 cytokine levels, ILC2 activation

  • Safety parameters: Impact on anti-helminth immunity, barrier integrity

  • Comparison with standard of care (corticosteroids, anti-IL-4/13)

This systematic approach will provide critical insights into the therapeutic window, efficacy parameters, and potential limitations of IL-17E antagonism as a treatment strategy for allergic diseases .

What are common technical challenges when working with recombinant IL-17E and their solutions?

When working with recombinant IL-17E, researchers may encounter several challenges:

Careful handling and quality control testing can mitigate most of these technical challenges.

How can researchers troubleshoot inconsistent responses in HEK-Blue IL-17 reporter assays?

For troubleshooting inconsistent HEK-Blue IL-17 reporter responses:

• Cell health and culture conditions:

  • Check cell viability (>90% viable cells required)

  • Maintain proper cell density (avoid overconfluence)

  • Ensure appropriate passage number (confirmed stable for 20 passages)

  • Verify selection antibiotic maintenance (Blasticidin, Hygromycin B, Zeocin)

• Assay execution variables:

  • Standardize incubation times (overnight stimulation)

  • Maintain consistent temperature (37°C) and CO₂ levels (5%)

  • Use freshly prepared QUANTI-Blue Solution

  • Control plate edge effects (use buffer in outer wells)

• Signal detection optimization:

  • Verify spectrophotometer calibration

  • Optimize reading wavelength (620-655 nm)

  • Ensure appropriate integration time

  • Consider kinetic reading to determine optimal timepoint

• Experimental controls:

  • Include positive control (hIL-17A at 10 ng/ml)

  • Include negative control (TNF-α at 10 ng/ml)

  • Run full dose-response curves

  • Test for receptor expression levels periodically

These systematic troubleshooting approaches can identify and address sources of variability in HEK-Blue IL-17 reporter assays .

What technical considerations should be addressed when analyzing IL-17E expression in complex tissue samples?

Analyzing IL-17E expression in complex tissues presents several technical challenges:

• Sample preparation optimization:

  • Fresh samples provide most reliable results

  • Standardize time from collection to processing

  • Use RNase inhibitors for RNA analysis

  • Consider mild fixation methods that preserve epitopes

• Detection method selection:

  • For mRNA: RNAscope offers cellular resolution with high sensitivity

  • For protein: Multiplex IHC with tyramide signal amplification

  • Single-cell approaches for heterogeneous tissues

  • Consider laser capture microdissection for region-specific analysis

• Specificity controls:

  • Include isotype controls for antibody-based methods

  • Use IL-17E knockout tissue as negative control when available

  • Perform blocking experiments with recombinant protein

  • Validate findings with orthogonal methods

• Quantification approaches:

  • Digital image analysis with machine learning algorithms

  • Colocalization with cell type-specific markers

  • Normalization to appropriate housekeeping genes/proteins

  • Relative quantification against standard curves

These considerations help ensure accurate detection and quantification of IL-17E in complex tissue environments where expression may be heterogeneous and often at low levels .

How can researchers effectively study IL-17E in the context of the gut-lung axis?

To investigate IL-17E's role in gut-lung immunological cross-talk:

• Dual-site sampling strategy:

  • Coordinate intestinal and lung tissue collection

  • Analyze matched BAL and intestinal lavage fluids

  • Include draining lymph nodes from both compartments

  • Consider time-course sampling for migration studies

• Microbiome influence assessment:

  • Compare germ-free, specific pathogen-free, and conventionally housed animals

  • Perform selective microbiota depletion using targeted antibiotics

  • Analyze metabolite profiles alongside IL-17E levels

  • Consider fecal microbiota transfer experiments

• Cell trafficking studies:

  • Use photoconvertible protein systems (Kaede mice) to track cell migration

  • Employ adoptive transfer of labeled IL-17E-responsive cells

  • Analyze shared lymphoid populations between compartments

  • Monitor inflammatory cell recruitment patterns

• Experimental perturbation approaches:

  • Compare airway vs. intestinal helminth infections

  • Study dual-site allergen challenges

  • Investigate how intestinal barrier disruption affects pulmonary IL-17E responses

  • Test compartment-specific IL-17E neutralization

These approaches help dissect the bidirectional communication between gut and lung immune systems, where IL-17E serves as a critical mediator in type 2 inflammation across mucosal barriers .

What are the methodological considerations for studying IL-17E in the context of epithelial barrier function?

For investigating IL-17E's impact on epithelial barrier integrity:

• Barrier function measurement techniques:

  • Transepithelial electrical resistance (TEER) for tight junction integrity

  • FITC-dextran permeability assays with multiple molecular weights

  • Immunofluorescence analysis of junction proteins (claudins, occludin, ZO-1)

  • In vivo barrier assessment using serum biomarkers of permeability

• Epithelial model selection:

  • Primary vs. immortalized epithelial cells

  • Air-liquid interface cultures for respiratory epithelium

  • Organoid models for three-dimensional architecture

  • Co-culture systems with immune cells

• Molecular mechanisms investigation:

  • Junction protein expression analysis (qPCR, Western blot)

  • Cytoskeletal organization assessment (F-actin staining)

  • Mucin production quantification (MUC5AC, MUC2)

  • Antimicrobial peptide expression

• Context-dependent modulation:

  • Examine IL-17E effects under basal vs. inflammatory conditions

  • Test barrier recovery after mechanical or chemical disruption

  • Compare acute vs. chronic IL-17E exposure

  • Investigate synergy with other epithelial-active cytokines (IL-13, TSLP)

These methodological approaches allow comprehensive evaluation of how IL-17E influences epithelial barrier function, which may contribute to its role in allergic inflammation and anti-helminth immunity .

How can transcriptomic approaches be optimized to study IL-17E-specific gene signatures?

For optimizing transcriptomic analysis of IL-17E-specific signatures:

• Experimental design considerations:

  • Include multiple timepoints (early: 2-6h, intermediate: 12-24h, late: 48-72h)

  • Dose-response series to identify threshold-dependent genes

  • Compare with other IL-17 family members to identify unique signatures

  • Include IL-17E antagonism conditions for validation

• Cell type optimization:

  • Use FACS-sorted primary cells when possible

  • Compare responses across different IL-17 receptor-expressing cells

  • Consider single-cell RNA-seq for heterogeneous populations

  • Include reference cell types for comparative analysis

• Bioinformatic analysis pipeline:

  • Pathway enrichment focusing on Th2 inflammation and tissue remodeling

  • Transcription factor binding site analysis for key regulators

  • Integration with epigenomic data (ATAC-seq, ChIP-seq)

  • Network analysis to identify gene regulatory hubs

  • Cross-reference with asthma and allergy GWAS datasets

• Validation approaches:

  • qPCR confirmation of key signature genes

  • Protein-level validation by Western blot or flow cytometry

  • Functional studies of identified targets

  • In vivo confirmation in relevant disease models

This comprehensive approach enables identification of robust IL-17E-specific transcriptional signatures that distinguish its function from other IL-17 family members and provide insights into its unique role in type 2 immune responses .